Field of the Invention
The present invention relates to an apparatus for analysing the random sequence of monomer units in a polymer, for example a biopolymer. It is especially useful in determining the sequence of nucleotides in naturally occurring polynucleotides such as RNA, DNA or synthetic analogues thereof.
Description of the Related Art
Next generation sequencing of genetic material is already making a significant impact on the biological sciences in general and medicine in particular as the unit cost of sequencing falls in line with the coming to market of faster and faster sequencing machines. For example, our co-pending application WO 2009/030953 discloses a new fast sequencer in which inter alia the sequence of nucleotides (bases or base pairs) in a single or double stranded nucleic acid sample (e.g. naturally occurring RNA or DNA) is read by translocating the same through a nano-perforated substrate provided with plasmonic structures juxtaposed within or adjacent the outlet of the nanopores. In this device, the plasmonic structures define detection windows within which each nucleotide (optionally labelled) is in turn induced to fluoresce or Raman scatter photons in characteristic way by interaction with incident light. The photons so generated are then detected remotely, multiplexed and converted into a data stream whose information content is characteristic of the nucleotide sequence itself. This sequence can then be recovered from the data stream using computational algorithms embodied in corresponding software programmed into a microprocessor integral therewith or in a computing device attached thereto.
Another device for fast sequencing nucleic acids is described for example in U.S. Pat. No. 6,627,067, U.S. Pat. No. 6,267,872 and U.S. Pat. No. 6,746,594. In its simplest form this device employs electrodes, instead of plasmonic structures, to define the detection window in or around the outlet of the nanopore. A potential difference is then applied across the electrodes and changes in an electrical property of the ionic medium flowing therebetween, as a consequence of the electrophoretic translocation of the nucleic acid sample and associated electrolyte therethrough, is measured as a function of time. In this device, as the various individual nucleotides constituting the nucleic acid pass through the detection window they continuously block and unblock it causing ‘blocking events’ which give rise to characteristic fluctuations in current flow or resistivity. These fluctuations are then used to generate a suitable data stream for analysis as described above.
One problem encountered with both types of device described above is the need to improve the number of nucleic acid molecules flowing through a given nanopore in a given detection interval or the effective utilisation of the total number of nanopores in the same interval as these parameters are directly related to both the signal to noise ratio characteristic of the detector's output and the ease with which the data stream can be accurately processed. Whilst this problem can in theory partly be offset by progressively multiplexing larger and larger numbers of nano-perforations in a given unit area of substrate, the practical problems associated with creating such a high density of nanopores means that it would be most desirable to find a method of improving efficiency levels at current densities.
WO2011/143340 describes a method for sequencing a nucleic acid involving the steps of (1) dissociating a plurality of optically labelled oligonucleotides (e.g. molecular beacons) from a labelled nucleic acid as molecules thereof translocate through an array of nanopores and detectors and (2) detecting optical signals from the displaced oligonucleotides. Whilst regulation of the speed of translocation of the labelled nucleic acid molecules is discussed, the provision of a means for specifically increasing the local concentration thereof adjacent the inlet of the nanopores is not specifically discussed.
WO2010/117470 discloses a nanopore sequencing device comprising an array of nanopores in a substrate connecting upper and lower fluidic regions which are in turned linked to upper and lower fluid volumes. The upper and lower fluid regions are connected to the upper and lower fluid volumes by means of resistive openings whose roles are to minimise electrical cross-talk between the detectors associated with each nanopore in the array by slowing down the rate of translocation of the nucleic acid. Typically the resistive openings are channels of cross-section narrower than that of the space defining the fluidic regions. This device is thus concerned with solving different problem to that claimed in our invention and accordingly does not include a means for increasing the local concentration of the analyte.
WO2011/040996 teaches an ultrafast nanopore sequencing device for sequencing nucleic acids whose nucleotides are provided with acceptor labels. It uses excitable donor labels, e.g. quantum dots, located within or adjacent the inlet or outlet of the pore, to energetically excite the acceptor labels by energy transfer so that they emit fluorescence which can be detected. The device does not include a means for increasing the local concentration of the nucleic acid.
Methods in Molecular Biology 385 9-12 (2007) discusses the functionalization of porous polymer monoliths using for example amines, so that they are able to concentrate and purify oligonucleotides. Methods of making such monoliths are disclosed as is their use in microfluidic chips including microchannels. There is no discussion of using such monoliths in nanopore sequencers for the purposes of solving the problem addressed by the present invention. Rather the concern is to improve the sample preparation steps associated with conventional biological chips.
Royal Society of Chemistry Special Publication 159-160 (2005) exemplifies the single-step concentration of DNA in a microfluidic channel. The method employed involves contacting a solution of two single-stranded, 12-mers DNA analytes with a solution of a DNA-poly(N,N-dimethylacrylamide) having differing affinities for the two. On the basis of this differing affinity the two analytes were separated by electrophoresis. This is a completely different approach to that used in our invention and appears to be concerned with product purification rather than sequencing.